Over the past year we have pursued studies on B cells in the setting of HIV disease by focusing on 1) mechanisms of immature/transitional B-cell expansion in HIV disease;2) changes in B-cell counts and subpopulations that occur following initiation of antiretroviral therapy (ART);3) changes in B-cell responses to HIV and recall antigens that occur following initiation of ART;and 4) mechanisms and modulation of HIV-associated B-cell exhaustion in HIV-viremic individuals and comparison with other disease settings. In the first study addressing mechanisms of immature/transitional B-cell expansion in the setting of HIV disease, we investigated changes in B-cell subpopulations that occurred in the peripheral blood of HIV-infected individuals who received the lymphocyte homeostatic cytokine IL-7 as part of a large safety trial. Our findings indicate that IL-7 itself can lead to the expansion of immature/transitional B cells in the peripheral blood. This is a novel observation given that a direct role for IL-7 on human B cells has never been established in vivo. This finding, along with results of a broader safety trial on the effects of IL-7 administration in HIV-infected individuals was recently published in the journal Blood. In ongoing follow-up studies, the roles of IL-7 and a more B-cell specific homeostatic factor, BAFF, are being investigated in the context of other disease parameters and changes in B-cell subpopulations that occur during HIV disease progression. In the second study, which is undergoing final statistical analyses, we investigated the changes in B cells and B-cell subpopulations that occur in individuals who initiate ART in the early stages of HIV infection and compared these findings to those we published last year for chronically-infected individuals in The Journal of Infectious Diseases. In early HIV-infected individuals we found a decrease in the number of B cells circulating in the blood compared to HIV-uninfected individuals and this decrease was reversed and normalized by ART. Furthermore, early HIV-infected individuals had lower frequencies of immature-transitional and exhausted B cells compared to chronically HIV-infected individuals, consistent with the association of these two B-cell subpopulations with advancing disease and chronic viremia respectively. In contrast, early HIV infection was associated with a higher frequency of plasmablasts compared to chronic HIV infection, likely a reflection of the high level of activation and terminal differentiation associated with the rapid dynamics of early infection. Finally, as with chronic HIV infection, abnormalities in the B-cell compartment were normalized in early HIV-infected individuals following effective ART. However, there were indications that the irreversible loss of memory B cells observed in chronic infection may be reversible in individuals who initiate ART during early infection. The third and fourth studies were undertaken following our publication in 2008 in The Journal of Experimental Medicine describing HIV-associated B-cell exhaustion. Similar findings have since been reported in several other disease settings that lead to chronic B-cell activation, including malaria. We collaborated on a malaria study that was recently published in the Journal of Immunology. In the third study, which is about to undergo final statistical analyses, we used a longitudinal approach to address the effect of ART on memory B-cell responses against HIV and recall antigen. The response to recall antigen was intended to measure overall B-cell function in individuals who successfully suppress HIV viremia with ART whereas HIV-specific B-cell responses following ART were measured to determine if this type of response is redirected to a stable memory compartment once HIV viremia is removed. We found that the reduction of HIV viremia by ART preserves and even increases memory B-cell responses to recall antigens such as the influenza vaccine. In contrast, the reduction of HIV viremia by ART led to a total disappearance of the HIV-specific memory B-cell responses in both early and chronic HIV-infected individuals, suggesting that the antibody response against HIV in infected individuals is difficult to maintain in the absence of ongoing viral replication. In the fourth study, a risky and difficult endeavor, we investigated the mechanism of B-cell exhaustion in HIV-infected individuals. We hypothesized that B-cell exhaustion was maintained by the high expression of multiple inhibitory receptors and spent several months devising an approach that would decrease the expression of these inhibitory receptors and enable us to evaluate the effect of this reduction on proliferation and cytokine secretion. The approach involved knocking down gene expression by siRNA transfection, something that has never been achieved with human primary B cells. We were nonetheless successful in applying this technique to the subpopulation of B cells in HIV-infected individuals that we had previously shown to be responsible for B-cell exhaustion and we found that knocking down the expression of several inhibitory receptors led to increased B-cell proliferation and secretion of effector B-cell cytokines. These seminal findings indicating that certain inhibitory receptors play an important role in B-cell exhaustion will soon be submitted publication.